4Pi STED fluorescence light microscope with high three-dimensional spatial resolution

Abstract

An apparatus forming an area of minimum light intensity enclosed by high light intensity includes two objectives focusing light of opposite directions into a common focal area. Light intensities of a first pair of light beams extinguish each other in a first partial area of the focal area. Beam paths of the first pair of beams pass through the objectives in a first pair of partial areas of the pupils of the objectives. Light intensities of a second pair of light beams extinguish each other in a second partial area of the focal area. Beam paths of the second pair of light beams pass through the objectives in a second pair of partial areas which are offset with regard to the first pair of partial areas of the pupils; and the light of the second pair does not interfere with the light of the first pair of light beams.

I claim: 
     
       1. An apparatus for forming a three-dimensional light intensity distribution comprising a spatially limited area of minimum light intensity that is enclosed by areas of higher light intensity, the apparatus comprising:
 two objectives
 which are facing each other on a common optical axis, 
 which focus light coming out of opposite directions into a common focal area, and 
 which each have a pupil, the common optical axis passing through a center of the pupil, 
 
 at least one light source, and 
 a first pair of beam paths each extending from the at least one light source, through one of the objectives and to the common focal area, wherein light intensities of a first pair of coherent light beams each coming from the at least one light source and running along one of the beam paths of the first pair of beam paths extinguish each other in a first partial area of the focal area by destructive interference as the two beam paths of the first pair of beam paths differ from each other in optical length by λ1 (2n+1)/2, wherein λ1 is the wavelength of the light of the first pair of coherent light beams and n is an integer, 
 wherein each of the beam paths of the first pair of beam paths pass passes through one of the two objectives in a first pair of partial areas of the pupils of the objectives centered on opposite sides of the common optical axis, 
 wherein the apparatus further comprises a second pair of beam paths each extending from the at least one or a second light source, through one of the objectives and to the common focal area, wherein light intensities of a second pair of coherent light beams each coming from the at least one or the second light source and running along one of the beam paths of the second pair of beam paths extinguish each other in a second partial area of the focal area by destructive interference as the two beam paths of the second pair of beam paths differ from each other in optical length by λ2 (2m+1)/2, wherein λ2 is the wavelength of the light of the second pair of coherent light beams and m is an integer, 
 wherein each of the beam paths of the second pair of beam paths passes through one of the two objectives in one of a second pair of partial areas of the pupils of the objectives centered on opposite sides of the common optical axis, 
 wherein the two partial areas of the second pair of partial areas of the pupils of the objectives are offset in a rotational direction about the common optical axis with regard to the two partial areas of the first pair of partial areas of the pupils of the objectives, and 
 wherein the light of the second pair of coherent light beams does not interfere with the light of the first pair of coherent light beams in the common focal area as the light of the second pair of coherent light beams and the light of the first pair of coherent light beams display at least one of the following features: they are non-coherent, their wavelengths are not identical, their polarizations are not identical, and they do not coincide in time. 
 
     
     
       2. The apparatus of  claim 1 , further comprising at least one further pair of beam paths each extending from the at least one or a further light source, through one of the objectives and to the common focal area, wherein light intensities of a further pair of coherent light beams each coming from the at least one or the further light source and running along one of the beam paths of the at least one further pair of beam paths extinguish each other in a further partial area of the focal area by destructive interference as the two beam paths of the at least one further pair of beam differ from each other in optical length by λ3 (2p+1)/2, where λ3 is the wavelength of the light of the further pair of coherent light beams and p is an integer,
 wherein each of the beam paths of the at least one further pair of beam paths passes through one of the two objectives in one of a first pair of partial areas of the pupils of the objectives centered on opposite sides of the common optical axis, 
 wherein the two partial areas of the further pair of partial areas of the pupils of the objectives are offset in the rotational direction about the common optical axis with regard to the two partial areas of all other pairs of partial areas of the pupils of the objectives, and 
 wherein the light of the further pair of coherent light beams does not interfere with the light of any other pair of coherent light beams in the common focal area as the light of the further pair of coherent light beams and the light of any other pair of coherent light beams display at least one of the following features: they are non-coherent, their wavelengths are not identical, their polarizations are not identical, and they do not coincide in time. 
 
     
     
       3. The apparatus of  claim 1 , wherein no partial area of the pupils of the objectives extends beyond the common optical axis, and/or wherein the partial areas of the pupil of each of the objectives through which the different beam paths pass towards the common focal area do not overlap. 
     
     
       4. The apparatus of  claim 1 , wherein the two partial areas of each pair of partial areas of the pupils of the objectives are point symmetric with regard to the common focal area. 
     
     
       5. The apparatus of  claim 1 , wherein the partial areas of the pupil of each of the objectives through which the different beam paths pass towards the common focal area are rotationally symmetric with regard to the common optical axis. 
     
     
       6. The apparatus of  claim 1 , wherein the two beam paths of the first pair of beam paths part from each other in a first beam splitter which splits light coming from the at least one light source for providing the first pair of coherent light beams. 
     
     
       7. The apparatus of  claim 6 , wherein the two beam paths of the second pair of beam paths part from each other in the first beam splitter which also splits light coming from the second light source for providing the second pair of coherent light beams. 
     
     
       8. The apparatus of  claim 7 , wherein the light from the at least one light source and the light from the second light source differ from each other in wavelength, and wherein the beam paths of the first and of the second pairs of beam paths include wavelength selective elements. 
     
     
       9. The apparatus of  claim 6 , wherein the two beam paths of the second pair of beam paths part from each other in a second beam splitter which splits light coming from the second light source for providing the second pair of coherent light beams. 
     
     
       10. The apparatus of  claim 6 , wherein the two beam paths of the second pair of beam paths part from each other in a second beam splitter which splits light coming from the first light source for providing the second pair of coherent light beams, and wherein an optical delay of a greater length than a coherence length or pulse length of the at least one light source is arranged between the at least one light source and the second beam splitter. 
     
     
       11. The apparatus of  claim 1 , wherein the two beam paths of the first and of the second pairs of beam paths part from each other in a common beam splitter which splits light coming from the at least one light source for providing the first and the second pairs of coherent light beams, and wherein the first and the second pairs of beam paths differ from each other in optical length by more than a coherence length or pulse length of the at least one light source. 
     
     
       12. The apparatus of  claim 1 , wherein the two beam paths of the first and of the second pairs of beam paths part from each other in a common beam splitter which splits light coming from the at least one light source for providing the first and the second pairs of coherent light beams, and wherein the first and the second pairs of coherent light beams differ from each other in polarization. 
     
     
       13. A scanning fluorescence light microscope comprising
 an apparatus for forming a three-dimensional light intensity distribution comprising a spatially limited area of minimum light intensity that is enclosed by areas of higher light intensity, the apparatus comprising:
 two objectives
 which are facing each other on a common optical axis, 
 which focus light coming out of opposite directions into a common focal area, and 
 which each have a pupil, the common optical axis passing through a center of the pupil, 
 
 at least one light source, and 
 a first pair of beam paths each extending from the at least one light source, through one of the objectives and to the common focal area, wherein light intensities of a first pair of coherent light beams each coming from the at least one light source and running along one of the beam paths of the first pair of beam paths extinguish each other in a first partial area of the focal area by destructive interference as the two beam paths of the first pair of beam paths differ from each other in optical length by λ1 (2n+1)/2, wherein λ1 is the wavelength of the light of the first pair of coherent light beams and n is an integer, 
 wherein each of the beam paths of the first pair of beam paths pass through one of the two objectives in one of a first pair of partial areas of the pupils of the objectives centered on opposite sides of the common optical axis, 
 wherein the apparatus further comprises a second pair of beam paths each extending from the at least one or a second light source, through one of the objectives and to the common focal area, wherein light intensities of a second pair of coherent light beams each coming from the at least one or the second light source and running along one of the beam paths of the second pair of beam paths extinguish each other in a second partial area of the focal area by destructive interference as the two beam paths of the second pair of beam paths differ from each other in optical length by λ2 (2m+1)/2, wherein λ2 is the wavelength of the light of the second pair of coherent light beams and m is an integer, 
 wherein each of the beam paths of the second pair of beam paths pass through one of the two objectives in one of a second pair of partial areas of the pupils of the objectives centered on opposite sides of the common optical axis, 
 wherein the two partial areas of the second pair of partial areas of the pupils of the objectives are offset in a rotational direction about the common optical axis with regard to the two partial areas of the first pair of partial areas of the pupils of the objectives, and 
 wherein the light of the second pair of coherent light beams does not interfere with the light of the first pair of coherent light beams in the common focal area as the light of the second pair of coherent light beams and the light of the first pair of coherent light beams display at least one of the following features: they are non-coherent, their wavelengths are not identical, their polarizations are not identical, and they do not coincide in time; 
 
 a detector detecting fluorescence light stemming from the common focal area and passed through at least one of the two objectives; and 
 a scanner for shifting the common focal area with regard to a sample stage. 
 
     
     
       14. The scanning fluorescence light microscope of  claim 13 , further comprising
 a fluorescence excitation device providing fluorescence excitation light which is focused by at least one of the two objectives to a focal point in the common focal area, 
 wherein the light of the second pair of coherent light beams and the light of the first pair of coherent light beams provided by the apparatus is fluorescence inhibiting light.